| Improving Phytase Enzyme Activity in a Recombinant phyA Mutant Phytase from Aspergillus niger N25 by Error-Prone PCR. | |
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MedLine Citation:
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PMID: 22101445 Owner: NLM Status: Publisher |
Abstract/OtherAbstract:
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The mutant acid phytase (phyA ( m )) gene was modified by random mutagenesis to improve enzymatic activity by using an error-prone PCR (ep-PCR) strategy. The mutated gene was linearized and inserted into plasmid vector pPIC9K and transformed by electroporation into Pichia pastoris GS115. A single transformant, PP-NP(ep)-6A, showing the strongest phytase activity from among the 5,500 transformants, was selected for detailed analyses. Southern blot analysis of the mutant yeast transformant showed that phyA ( ep ) gene was integrated into the chromosome genome through single crossover with one copy of phyA. The kinetic parameters indicated that the mutant one showed 61% higher specific activity and 53% lower k (m) value than that of PP-NP(m)-8 (P < 0.05). In addition, the overall catalytic efficiency (k (cat)/k (m)) of the mutant one was 84% higher (P < 0.05) than that of PP-NP(m)-8. Nine bases were altered in the mutant sequences, which resulted in three amino acid changes, namely, Glu156Gly, Thr236Ala, and Gln396Arg. The structural predictions indicated that the mutations generated by ep-PCR somehow reorganized or remodeled the active site, which could lead to increasing catalytic efficiency. |
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Authors:
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Yan Liao; Min Zeng; Zhen-Fang Wu; Hui Chen; Hong-Ning Wang; Qi Wu; Zhi Shan; Xue-Yi Han |
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Publication Detail:
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Type: JOURNAL ARTICLE Date: 2011-11-19 |
Journal Detail:
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Title: Applied biochemistry and biotechnology Volume: - ISSN: 1559-0291 ISO Abbreviation: - Publication Date: 2011 Nov |
Date Detail:
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Created Date: 2011-11-21 Completed Date: - Revised Date: - |
Medline Journal Info:
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Nlm Unique ID: 8208561 Medline TA: Appl Biochem Biotechnol Country: - |
Other Details:
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Languages: ENG Pagination: - Citation Subset: - |
Affiliation:
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College of Life and Physical Science, Sichuan Agricultural University, Ya'an, 625014, China. |
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From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine
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